Device and method for separating plasma from a biological fluid

ABSTRACT

A device and method for processing a biological fluid comprises directing the biological fluid tangentially or parallel to the face of a separation medium in at least one serpentine fluid flow channel such that a plasma-rich fluid passes through the separation medium and a plasma-depleted fluid passes tangentially to the surface of the separation medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase filing under 35 U.S.C. §371 ofPCT/US92/09542, filed Nov. 3, 1992, and published as WO93/08904 May 13,1993, which is a continuation-in-part of U.S. patent application Ser.No. 07/787,067, filed Nov. 4, 1991, now abandoned.

TECHNICAL FIELD

The present invention concerns a device and method for separating plasmafrom a biological fluid such as blood.

BACKGROUND OF THE INVENTION

An adult human contains about 5 liters of blood, of which red bloodcells account for about 45% of the volume, white cells about 1%, and thebalance being liquid blood plasma. Blood also contains large numbers ofplatelets. In view of the substantial therapeutic and monetary value ofblood components such as red blood cells, platelets, and plasma, avariety of techniques have been developed to separate blood into itscomponent fractions while ensuring maximum purity and recovery of eachof the components.

Typically, donated blood is collected in a blood collection bag andseparated by centrifugation into PRC and platelet-rich plasma (PRP)fractions, the latter of which is in current practice separated by asecond centrifugation to provide plasma and PC. With respect to thesecond centrifugation step, the platelet concentrate is typicallyobtained from the PRP by "hard-spin" centrifugation (rotating at about5000 G). This hard-spin compacts the platelets into a pellet orconcentrate at the bottom of the test tube, flask, or bag. The plasmacomponent is then removed or expressed to a separate bag or container,leaving the platelet component and some plasma behind. This plateletcomposition, which tends to form a dense aggregate, is subsequentlydispersed to make PC. The dispersion step is usually carried out bygentle mixing, for example, by placing the bag on a moving table whichrotates with a precessing tilted motion.

Platelet concentrate can also be prepared using apheresis of autologousblood. With this method, whole blood is removed from a single donor, andcentrifuged into its component parts. The desired component is thenharvested and the remainder of the blood is returned to the donor. Thisprocedure allows collection of multiple units from one donor. Typically,a 2 to 3 hour apheresis procedure will produce a platelet productcontaining 3×10¹¹ platelets, equivalent to about six to ten units ofrandom donor platelets, i.e., a typical transfusion unit. The commonpractice with respect to platelet concentrate is to transfuse a pool ofsix to ten units of platelets per administration, containing a total ofabout 300 to 700 ml of platelet concentrate.

Blood bank personnel have responded to the increased need for bloodcomponents by attempting to increase packed red cell (PRC) and plateletconcentrate (PC) yields in a variety of ways. For example, in separatingthe PRP from PRC, blood bank personnel have attempted to ensure that theentire PRP fraction is recovered, but this may be counter-productive,since the PRP, and the PC subsequently extracted from it, are frequentlycontaminated by red cells, giving a pink or red color to the normallylight yellow PC. The presence of red cells in PC is so highlyundesirable that pink or red PC is frequently discarded, or subjected torecentrifugation, both of which increase operating costs and are laborintensive.

Additionally, freshly donated blood contains platelets varying in agefrom newly-formed to 9 days or more in age. Newly-formed platelets arelarger, and are generally believed to be more active. Because theyounger platelets are larger, they tend to sediment faster during thefirst centrifugation step, and consequently are present in largernumbers in the PRP nearest to the red cell interface. Thus, although itis desirable to reclaim a larger proportion of the younger, more activeplatelets, attempting to obtain a greater quantity poses a risk ofcontamination with red cells.

Typical techniques for processing platelets or platelet concentrate mayreduce the yield and/or adversely affect the platelets. For example, asnoted earlier, during the separation of PRP from PRC, it is difficult toefficiently obtain the maximum yield of platelets while preventing redcells from entering the plasma. Additionally, hard spin centrifugationand dispersion is labor intensive and it is believed that the forcesapplied during centrifugation may damage the platelets. For example, thehard-spin is potentially damaging to the platelets as it induces partialactivation agglomeration of the platelets and may cause physiologicaldamage. Such agglomeration requires several hours to resuspend theplatelets in solution before they can be used for transfusion into apatient. Additionally, the several hour dispersion step is anundesirable delay, and is believed by many researchers to partiallyaggregate the platelet concentrate.

Furthermore, the hard-spin typically produces "distressed" plateletswhich partially disintegrate upon resuspension. Unfortunately, whilemixing does prevent agglomeration, it encourages gas exchange bydiffusion of oxygen through the walls of the bag (thereby controllingpH), and bathes the product in needed nutrients, this requires time,resulting in an increase in the number and size of microaggregates.Further, over time, gel-like bodies may be formed, which may comprisefibrinogen, degenerated protein, and degenerated nucleic acids, whichmay interfere with the separation of the platelets from the donatedblood. Thus, some platelets are lost due to the process conditions.

Additionally, since platelets are notorious for being "sticky", anexpression reflecting the tendency of platelets suspended in bloodplasma to adhere to any non-physiological surface to which they areexposed, the recovery of platelets may be adversely affected during thepreparation of platelet concentrate, regardless of the method ofpreparation. Furthermore, under many circumstances, platelets alsoadhere strongly to each other. Accordingly, in recovering platelets, itis desirable to restrict platelet loss to about 15% or less of theoriginal platelet concentration.

Moreover, while leukocyte depletion of blood components for transfusionmay decrease risk to the patient, when leukocytes are removed from, forexample, platelet-rich plasma, which typically results in the productionof a leukocyte-free platelet concentrate, the platelet component of thefiltrate usually passes through a filter or separation device. In thesesystems, platelets may adhere to the surfaces of components of theseparation device; such adhesion tends to cause substantial, andsometimes complete, removal of platelets from the filtrate. Furthermore,platelet concentrate present within the separation device at thecompletion of the separation process will be lost.

DISCLOSURE OF INVENTION

In describing the present invention, the following terms are used asdefined below.

(A) Biological Fluid:

Biological fluids include any treated or untreated fluid associated withliving organisms, particularly blood, including whole blood, warm orcold blood, and stored or fresh blood; treated blood, such as blooddiluted with at least one physiological solution, including but notlimited to saline, nutrient, and/or anticoagulant solutions; bloodcomponents, such as platelet concentrate (PC), platelet-rich plasma(PRP), platelet-poor plasma (PPP), platelet-free plasma, plasma, packedred cells (PRC), or buffy coat (BC); analogous blood products derivedfrom blood or a blood component or derived from bone marrow; red cellsseparated from plasma and resuspended in physiological fluid; andplatelets separated from plasma and resuspended in physiological fluid.The biological fluid may include leukocytes, or may be treated to removeleukocytes. As used herein, blood product or biological fluid refers tothe components described above, and to similar blood products orbiological fluids obtained by other means and with similar properties.

A "unit" is the quantity of biological fluid from a donor or derivedfrom one unit of whole blood. It may also refer to the quantity drawnduring a single donation. Typically, the volume of a unit varies, theamount differing from patient to patient and from donation to donation.Multiple units of some blood components, particularly platelets andbuffy coat, may be pooled or combined, typically by combining four ormore units.

B) Plasma-Depleted Fluid:

A plasma-depleted fluid refers to a biological fluid which has had somequantity of plasma-rich fluid (defined below) removed therefrom, e.g.,the platelet-rich fluid or platelet component obtained when plasma isseparated from PRP, or the fluid which remains after plasma is removedfrom whole blood. The separation of the plasma-rich fluid from thebiological fluid produces a plasma-depleted fluid having an increasedconcentration of platelets and/or red cells on a volume basis.Typically, the plasma-depleted fluid is a platelet-containing fluid.

C) Plasma-Rich Fluid:

A plasma-rich fluid refers to the plasma portion or plasma componentremoved from a biological fluid, e.g., the plasma-rich fluid when plasmais separated from PRP, or the plasma which is removed from whole blood.The plasma-rich fluid separated from a biological fluid has an increasedconcentration of plasma on a volume basis. Typically, the plasma-richfluid is the plasma-containing fluid that passes through a separationmedium. Exemplary plasma-rich fluids include platelet-poor plasma orplatelet-free plasma.

D) Separation medium:

A separation medium refers to a porous medium through which one or morebiological fluids pass and which separates one component of thebiological fluid from another. As noted in more detail below, the porousmedium for use with a biological fluid may be formed from any natural orsynthetic fiber or from a porous or permeable membrane (or from othermaterials of similar surface area and pore size) compatible with abiological fluid, typically a biological fluid containing platelets,e.g., whole blood or PRP. The surface of the fibers or membrane may beunmodified or may be modified to achieve a desired property.

Although the separation medium may remain untreated, the fibers ormembrane are preferably treated to make them even more effective forseparating one component of a biological fluid, e.g., plasma, from othercomponents of a biological fluid, e.g., platelets or red cells. Theseparation medium is preferably treated in order to reduce or eliminateplatelet adherence to the medium. Any treatment which reduces oreliminates platelet adhesion is included within the scope of the presentinvention. Furthermore, the medium may be surface modified in order toachieve a desired critical wetting surface tension (CWST), e.g., asdisclosed in U.S. Pat. Nos. 4,880,548 and 5,100,564, and InternationalPublication No. WO 92/07656 in order to increase the critical wettingsurface tension (CWST) of the medium and to be less adherent ofplatelets. Defined in terms of CWST, a preferred range of CWST for aseparation medium as provided by the present invention is above about 53dynes/cm, typically above about 70 dynes/cm. The CWST of the separationmedium may be dictated by its intended use. Further, the medium may besubjected to gas plasma treatment, an exemplary purpose for which is toreduce platelet adhesion.

The porous medium may be pre-formed, single or multi-layered, and/or maybe treated to modify the surface of the medium. If a fibrous medium isused, the fibers may be treated either before or after forming thefibrous lay-up. It is preferred to modify the fiber surfaces beforeforming the fibrous lay-up because a more cohesive, stronger product isobtained after hot compression to form an integral element.

The separation medium may be configured in any suitable fashion, such asa flat sheet, a composite of two or more layers, a corrugated sheet, aweb, hollow fibers, or a membrane.

F) Tangential flow filtration:

As used herein, tangential flow filtration refers to passing orcirculating a biological fluid in a generally parallel or tangentialmanner to the surface of the separation medium.

The present invention provides a method for processing a biologicalfluid comprising: directing a biological fluid tangentially to thesurface of a separation medium whereby plasma-rich fluid passes throughthe separation medium and plasma-depleted fluid passes tangentiallyacross the separation medium.

The present invention provides a device for removing plasma from abiological fluid comprising: a housing having an inlet and first andsecond outlets and defining a first liquid flow path between the inletand the first outlet and a second liquid flow path between the inlet andthe second outlet; and a separation medium positioned inside the housingtangentially to the first flow path and across the second flow path, theseparation medium being suitable for passing plasma therethrough.

The present invention provides a device for processing a biologicalfluid comprising: a housing having a first portion and a second portion;an inlet and a first outlet in the first portion and a first fluid flowpath therebetween; a second outlet in the second portion and a secondfluid flow path between the inlet and the second outlet; and aseparation medium positioned inside the housing between the firstportion and the second portion and tangentially to the first flow pathand across the second flow path, the separation medium being suitablefor passing plasma therethrough but not platelet-rich fluid.

The present invention provides a device for treating a biological fluidcomprising: a separation medium having first and second externalsurfaces and being suitable for passing plasma therethrough; and ahousing defining first and second flow paths, the separation mediumbeing disposed within the housing wherein the first flow path extendstangentially to the first external surface of the separation medium andthe second flow path extends from the first external surface through theseparation medium to the second external surface.

The present invention provides for a system for processing a biologicalfluid comprising: a housing having an inlet and first and second outletsand defining a first liquid flow path between the inlet and the firstoutlet and a second liquid flow path between the inlet and the secondoutlet; a separation medium positioned inside the housing tangentiallyto the first flow path and across the second flow path, the separationmedium being suitable for passing a plasma-rich fluid therethrough, anda container in fluid communication with the second outlet. The systemmay also include another container in fluid communication with the firstoutlet.

The invention involves the treatment of a biological fluid tonon-centrifugally separate at least one component from the biologicalfluid, e.g., treating PRP to obtain plasma and PC, or separating plasmafrom whole blood. Processes and devices provided by the inventionutilize a separation medium that allows the passage of plasma, butprevents passage of platelets and/or red cells, through the medium,thereby eliminating the need for "hard-spin" centrifugation or multiplecentrifugations as processing steps. Tangential flow of a biologicalfluid parallel to the upstream surface of the separating medium permitsthe passage of plasma through the medium, while reducing the tendencyfor cellular components or platelets to adhere to the surface of themedium, thus assisting in the prevention of passage of platelets throughthe separation medium. The hydrodynamics of flow parallel to a surfaceare indeed believed to be such that during flow parallel to the surface,platelets develop a spin which causes them to be recovered from thesurface.

The device and method of the present invention thus protect plateletsand red blood cells from physiological damage, and directly andeffectively minimize or eliminate loss or damage caused by the currentlyused centrifugal separation processes, by reducing or eliminating theexposure to harmful centrifugation. Furthermore, the platelets and/orred blood cells are not required to pass through yet another filtrationdevice in order to be separated from PRP. A feature of the separationdevice of the invention, therefore, is the increased yield of clinicallyand therapeutically superior platelet concentrate and/or platelet-free(or platelet-poor) plasma.

Advantageous features of the devices and methods of the presentinvention include the separation of at least one component of abiological fluid from the rest of the fluid with minimal loss oractivation of platelets. Platelet function is believed to be onlyminimally affected by the separation process, and platelet survival timewithin the patient is believed to be significantly longer. Further,because of the high cost and increased demand for both plateletpreparations and for plasma, as well as the clinical need to deliver amaximum therapeutic dose, a device as provided by this invention candeliver a higher proportion of the platelets or plasma originallypresent in the sample. The present invention also provides forreclaiming a larger proportion of the younger, more active platelets ina sample.

This invention also provides for a device and method for separatingplatelet-poor plasma or platelet-free plasma from a biological fluid,such as PRP or from whole blood, without requiring rotation, spinning,or centrifugation to effect the separation. For example, the instantinvention provides for the separation of plasma from whole blood withoutcentrifugation. Additionally, the present invention provides forprocessing of PRP to form PC and plasma without hard-spincentrifugation.

The present invention further provides for maximum recovery of plasmafrom whole blood or from PRP.

Additionally, since some currently used procedures for processingbiological fluid require several hours for completion, the presentinvention reduces processing time.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elevation of an embodiment of the present invention.

FIG. 2 is an elevation of another embodiment of the present invention.

FIG. 3 is a cross-section of FIG. 2, along V--V, showing the first fluidflow path in a separation device as provided by the invention.

FIG. 4 is a section of FIG. 3, along I--I.

FIG. 5 is a section of FIG. 3, along II--II.

FIG. 6 is a cross-section of an embodiment of the invention showing thesecond fluid flow path in a separation device as provided by theinvention.

FIG. 7 is a section of FIG. 6, along III--III.

FIG. 8 is a section of FIG. 6, along IV--IV.

MODES FOR CARRYING OUT THE INVENTION

The present invention involves the separation of one or more componentsfrom a biological fluid. As provided by the present invention, abiological fluid, particularly blood, is exposed to a separation mediumsuitable for passing plasma therethrough, but not platelets and/or redcells. Clogging of the separation medium by platelets and/or red cellsis minimized or prevented.

The device of the present invention may be incorporated into a systemwhich includes one or more containers in fluid communication with theinlet and/or outlets of the separation device.

Exemplary biological fluid processing systems, which may be closedand/or sterile systems, are shown in FIGS. 1 and 2. Biological fluidprocessing system 100 may include a first container such as a collectionbag or syringe 19; a separation device 200 including a separation medium16; a second container (first satellite bag) 18; a third container(second satellite bag) 17. The processing system 100 may also include atleast one functional biomedical device, for example, a pump 90, and/orother functional biomedical devices, including filtration and/orseparation devices (not shown).

The components of the biological fluid processing system may be in fluidcommunication through conduits. For example, as illustrated in FIG. 1,conduits 50A, 50B, and 50C may be used to provide fluid communicationbetween the components of the system. The biological fluid processingsystem may also include a seal, valve, clamp, transfer leg closure,stopcock, or the like located within or on at least one of the conduitsand/or the containers.

As illustrated in FIG. 2, a separation device as provided by theinvention generally comprises a housing 10 which includes an inlet 11and first and second outlets 12 and 13, respectively; a first fluid flowpath 14 between the inlet 11 and the first outlet 12; and a second fluidflow path 15 between the inlet 11 and the second outlet 13. A separationmedium 16 having first and second surfaces 16a, 16b is positioned insidethe housing 10, the separation medium being positioned parallel to thefirst fluid flow path 14 and across the second fluid flow path 15.

As depicted in FIG. 2, in a separation device 200, the housing 10includes first and second portions 10a and 10b, and the separationmedium 16 is positioned inside the housing 10 between the first andsecond housing portions 10a, 10b. The first and second housing portions10a, 10b may be joined in any convenient manner, for example, byultrasonic or heat welding, an adhesive, a solvent, or one or moreconnectors.

Each of the components of the invention will now be described in moredetail below.

Embodiments of the present invention may be configured in a variety ofways to ensure maximum contact of the biological fluid with the firstsurface 16a of separation medium 16 and to reduce or eliminate cloggingon the first surface 16a of the separation medium. For example, theseparation device includes one or more channels, grooves, conduits,passages, or the like which may be serpentine, parallel, curved, or avariety of other configurations facing the first surface 16a of theseparation medium 16. Alternatively, the separation device may include afirst shallow chamber facing the first surface 16a of the separationmedium 16. The first chamber may include an arrangement of ribs whichspread the flow of biological fluid over the entire first surface 16a ofthe separation medium 16.

The fluid flow channels may be of any suitable design and construction.For example, the channels may have a rectangular, triangular, orsemi-circular cross section and a constant depth and width. Preferably,the channels have a rectangular cross section and vary in depth, forexample, between inlet 11 and outlet 12.

In the embodiment shown in FIGS. 3, 4, and 5, the inlet 11 of thehousing 10 is connected to serpentine fluid flow channels 20, 21, and 22which face the first surface 16a of the separation medium 16. Thesechannels 20-22 separate the inlet flow of biological fluid into separateflow paths tangential to the first surface 16a of the separation medium16. Extending along the first surface 16a, the serpentine fluid flowchannels 20, 21, and 22 may be recombined at first outlet 12 of thehousing 10.

Embodiments of the present invention may also be configured in a varietyof ways to minimize back pressure across the separation medium 16 and toensure a sufficiently high velocity of flow to the second outlet 12 toprevent fouling of surface 16a, while minimizing hold-up volume. Theseparation device may include an arrangement of ribs or may comprise oneor more channels, grooves, conduits, passages, or the like which may beserpentine, parallel, curved, or have a variety of other configurationsfacing the second surface 16b of the separation medium. Alternatively,the separation device may include a second shallow chamber facing thesecond surface 16b of the separation medium 16.

The fluid flow channels may be of any suitable design and construction.For example, the channels may have a rectangular, semi-circular, ortriangular cross section and a constant or variable depth and/or width.In the embodiment shown in FIGS. 6-8, several serpentine fluid flowchannels 31, 32, 33, 34, and 35 face the second surface 16b of theseparation medium 16. Extending along the second surface 16b, theserpentine fluid flow channels 31-35 may be recombined at the secondoutlet 13.

Ribs, walls, or projections 41, 42, 43, 44, and 45 may be used to definethe channels 20-22, 31-35 of the first and second chambers and/or maysupport or position the separation medium 16 within the housing 10. In apreferred embodiment of the invention, there are more walls in thesecond chamber than in the first chamber to prevent deformation of theseparation medium 16 caused by pressure differential through theseparation medium.

An exemplary channel depth may be in the range from about 0.635 cm(about 0.250 inch) to about 0.0025 cm (about 0.001 inch). An exemplarychannel width may be in the range from about 0.635 cm (about 0.250 in)to about 0.025 cm (about 0.010 inch).

The housing and the separation medium of the present inventive devicemay be of any suitable configuration and material. For example, thehousing, including the channels, ribs, walls, and/or projections may beformed from a material that is substantially impermeable to thebiological fluid and substantially unreactive with the biological fluid.In the illustrated embodiment, the channels are defined by three sideswhich are substantially impermeable to and substantially unreactive withthe biological fluid, and one side, i.e., defined by the separationmedium, that is permeable to the biological fluid. Alternatively, thechannels may be defined by two substantially impermeable andsubstantially unreactive sides and two permeable sides. For example, inone configuration, the opposing sides of a channel may each face aseparation medium, allowing plasma-rich fluid to flow through eachseparation medium, and plasma-depleted fluid to flow tangentially toeach separation medium. In another variation, e.g., involving ahalf-round configuration, at least one side of the channel issubstantially impermeable to and substantially unreactive with thebiological fluid.

While the preferred device has one inlet and two outlets, otherconfigurations can be employed without adversely affecting the properfunctioning of the device. For example, multiple inlets for a biologicalfluid may be used so long as the biological fluid flows tangentially tothe face of the separation medium. Alternatively, a single inlet and asingle outlet may be used. For example, a separation device may beconfigured to provide for two liquid flow paths such that both liquidflow paths communicate with the inlet, but only one liquid flow pathcommunicates with both the inlet and the outlet.

The separation medium may be arranged in the separation device in anysuitable manner so long as the biological fluid flow tangential orparallel to the separation medium is maintained to a sufficient extentto avoid or minimize substantial platelet adhesion to the separationmedium. One skilled in the art will recognize that platelet adhesion maybe controlled or affected by manipulating any of a number of factors:velocity of the fluid flow, configuration of the channel, depth and/orwidth of the channel, varying the depth and/or varying the width of thechannel, the surface characteristics of the separation medium, thesmoothness of the medium's surface, and/or the angle at which the fluidflow crosses the face of the separation medium, among other factors. Forexample, the velocity of the first fluid flow is sufficient to removeplatelets from the surface of the separation medium. Without intendingto be limited thereby, a velocity in excess of about 30 cm/second hasbeen shown to be adequate.

The velocity of the fluid flow may also be affected by the volume of thebiological fluid, by varying the channel depth, and by varying thechannel width. For example, as shown in FIG. 4, the channel depth may bevaried from about 0.635 cm (about 0.250 in) in the region 23 near theinlet 11 to about 0.0025 cm (about 0.001 in) in the region 24 near theoutlet 12. One skilled in the art will recognize that a desired velocitymay be achieved by manipulating these and other elements. Also,platelets may not adhere as readily to a separation medium having asmooth surface as compared to a medium having a rougher surface.

A separation medium, as provided by the present invention, comprises aporous medium suitable for passing plasma-rich fluid therethrough. Theseparation medium, as used herein, may include but is not limited topolymeric fibers (including hollow fibers), polymeric fiber matrices,polymeric membranes, and solid porous media. Separation media inaccordance with the invention separate plasma from a biological fluidcontaining platelets, typically whole blood or PRP, without allowing asubstantial amount of platelets and/or red cells to pass therethrough.

A separation medium according to the invention preferably exhibits anaverage pore rating generally or intrinsically smaller than the averagesize of platelets. Preferably, platelets do not adhere to the surface ofthe separation medium, thus reducing pore blockage. The separationmedium should also have a low affinity for proteinaceous components inthe biological fluid such as PRP or whole blood. This enhances thelikelihood that the plasma-rich fluid, e.g., platelet-free plasma, willexhibit a normal concentration of proteinaceous clotting factors, growthfactors, and other needed components. The separation medium and devicealso enhances the likelihood that complement activation will be avoided.

In accordance with the invention, a separation medium formed of fibersmay be continuous, staple, or melt-blown. The fibers may be made fromany material compatible with a biological fluid containing platelets,e.g., whole blood or PRP, and may be treated in a variety of ways tomake the medium more effective. Also, the fibers may be bonded, fused,or otherwise fixed to one another, or they may simply be mechanicallyentwined. A separation medium, as the term is used herein, may refer toone or more porous polymeric sheets, such as a woven or non-woven web offibers, with or without a flexible porous substrate, or may refer to amembrane formed, for example, from a polymer solution in a solvent byprecipitation of a polymer when the polymer solution is contacted by asolvent in which the polymer is not soluble. The porous, polymeric sheetwill typically be microporous, e.g., having a substantially uniform,continuous matrix structure containing a myriad of small largelyinterconnected pores.

The separation medium of this invention may be formed, for example, fromany synthetic polymer capable of forming fibers or a membrane. While notnecessary to the apparatus or method of the invention, in one variationthe polymer is capable of serving as a substrate for grafting withethylenically unsaturated monomeric materials. In this variation, thepolymer should be capable of reacting with at least one ethylenicallyunsaturated monomer under the influence of ionizing radiation or otheractivation means without the matrix being adversely affected. Suitablepolymers for use as the substrate include, but are not limited to,polyolefins, polyesters, polyamides, polysulfones, polyarylene oxidesand sulfides, and polymers and copolymers made from halogenated olefinsand unsaturated nitriles. Preferred polymers are polyolefins,polyesters, and polyamides, e.g., polybutylene terephthalate (PBT) andnylon. In an embodiment, a polymeric membrane may be formed from afluorinated polymer such as polyvinylidene difluoride (PVDF). The mostpreferred separation media are a microporous polyamide membrane or apolycarbonate membrane.

Exemplary separation media include but are not limited to thosedisclosed in International Publication No. WO 92/07656 and U.S. Pat.Nos. 4,886,836; 4,906,374; 4,964,989; 4,968,533; and 5,019,260, whichmay include separation media having a water permeability of up to about0.023 L/min/Pa/m² (about 15.0 L/min/psid/ft²).

Surface characteristics of a fiber or membrane can be modified by anumber of methods, for example, by chemical reaction including wet ordry oxidation, by coating the surface through deposition of a polymerthereon, by grafting reactions which are activated by exposure to anenergy source such as heat, a Van der Graff generator, ultravioletlight, or to various other forms of radiation, and by treatment of thefibers or membrane with a gas plasma. A typical method for treatmentwith a gas plasma utilizes radio frequency (RF) discharge, with orwithout one or more polymerizable species. A typical method for agrafting reaction uses gamma-radiation, for example, from a cobaltsource.

Radiation grafting, when carried out under appropriate conditions, hasthe advantage of considerable flexibility in the choice of reactants,surfaces, and in the methods for activating the required reaction.Gamma-radiation grafting is particularly preferable because the productsare very stable and have undetectably low aqueous extractable levels.Furthermore, the ability to prepare synthetic organic fibrous mediahaving a CWST within a desired range is more readily accomplished usinga gamma radiation grafting technique.

An exemplary radiation grafting technique employs at least one of avariety of monomers each comprising an ethylene or acrylic moiety and asecond group, which can be selected from hydrophilic groups (e.g.,--COOH, or --OH) or hydrophobic groups (e.g., a methyl group orsaturated chains such as --CH₂ CH₂ CH₃). Grafting of the fiber ormembrane surface may also be accomplished by compounds containing anethylenically unsaturated group, such as an acrylic moiety, combinedwith a hydroxyl group, such as, hydroxyethyl methacrylate (HEMA). Use ofHEMA as the monomer contributes to a very high CWST. Analogues withsimilar characteristics may also be used to modify the surfacecharacteristics of fibers.

In a variation of the invention, the separation medium is surfacemodified by grafting thereon a hydroxyl-containing monomer to provide aseparation medium having a low affinity for proteinaceous substances.For example, as described in U.S. Pat. No. 4,906,374, the separationmedium, which is preferably a skinless membrane, may be surface modifiedusing hydroxyl-containing unsaturated monomers, more typicallymonofunctional unsaturated monomers rich in pendant hydroxyl groups orgroups capable of reacting to form hydroxyl groups, which are capable ofundergoing polymerization and covalently bonding to the substrate underthe influence of ionizing radiation. The most preferredhydroxyl-containing monomers are those in which the hydroxyl group ispendant, i.e., the group is not attached to a carbon atom which formspart of the polymer's backbone but is bound to a carbon atom that isseparated from the backbone as, for example, a branching carbon atom.Suitable monomeric compounds should be substantially completely, if nottotally, soluble in the solvents used. Solutions of the monomer compoundmay range in concentration of the monomer(s) from about 0.1 to about 5.0percent, by weight, preferably about 0.2 to about 3.0 percent, byweight, based on the total weight of the solution.

In another variation of the invention, the separation medium is treatedwith a gas plasma, typically a low temperature gas plasma, with orwithout deposition of a polymeric substance formed by the plasma orintroduced into the plasma. The term "plasma" or "gas plasma" is usedgenerally to describe the state of an ionized gas. The use of the term"plasma" in this context should not be confused with "plasma" as itrefers to a biological fluid. A gas plasma consists of high energycharged ions (positive or negative), electrons, and neutral species. Asknown in the art, a plasma may be generated by combustion, flames,physical shock, or, preferably, by electrical discharge, such as acorona or glow discharge.

In an exemplary gas plasma treatment technique, radio frequency (RF)discharge, a separation medium to be treated is placed in a vacuumchamber and the chamber is evacuated. Gas at low pressure is bled intothe system through the gas inbleed until the desired gas pressuredifferential across the conduit is achieved. An electromagnetic field isgenerated by subjecting the gas to a capacitive or inductive RFelectrical discharge. The gas absorbs energy from the electromagneticfield and ionizes, producing high energy particles. The gas plasma, asused in the context of the present invention, is exposed to theseparation medium, thereby modifying the properties of the medium toprovide it with characteristics not possessed by the untreated medium,e.g., improving its biocompatibility, and ability to reduce plateletadhesion.

The gas used to treat the surface of the medium may include inorganicand organic gases used alone or in combination according to need. Inaddition, the gas may be a vaporized organic material, such as anethylenic monomer to be plasma polymerized or deposited on the surfaceof the fiber. One example of a suitable gas is oxygen.

Typical parameters for treatment with a gas plasma may include powerlevels from about 10 to about 3000 watts. The RF frequency may includeabout 1 kHz to about 100 MHz. Exposure times may include about 5 secondsto about 12 hours. The gas pressures may include about 0.001 to 100torr; and a gas flow rate of about 1-2000 standard cc/min.

In accordance with the invention, the separation medium may be surfacemodified, typically by radiation grafting or gas plasma treatment, inorder to achieve the desired performance characteristics, wherebyplatelets are concentrated with a minimum of medium blocking. It mayalso be desirable to surface modify the separation medium such that theresulting plasma solution contains essentially all of its nativeproteinaceous constituents. Exemplary membranes having a low affinityfor proteinaceous substances are disclosed in U.S. Pat. Nos. 4,886,836;4,906,374; 4,964,989; 4,968,533; and 5,019,260.

Suitable membranes in accordance with an embodiment of the invention maybe skinless microporous membranes and may be produced by, for example, asolution casting method.

For the separation of about one unit of whole blood, a typicalseparation device as provided by the invention includes an effectivepore size smaller than platelets on the average, typically less thanabout 4 micrometers, preferably less than about 2 micrometers.

A typical separation device as provided by the invention includes aseparation medium having an effective surface area in the range of about1.94 cm² to about 194 cm² (about 0.3 in² to about 30 in²). As usedherein, the term effective surface area refers to the surface areacontacted by the biological fluid.

A preferable ratio of the wetted surface area of the fluid flow channelto the volume of the channel (A/V) is in the range of about 6.3 cm⁻¹ toabout 866 cm⁻¹ (about 16 in⁻¹ to about 2,200 in⁻¹).

The permeability of the separation medium is sufficient to allow thepassage of a desirable amount of a fluid therethrough at a reasonablepressure in a reasonable amount of time. With respect to biologicalfluid, a preferred permeability is in the range of from about 0.00078L/min/Pa/m² to about 0.023 L/min/Pa/m² (about 0.5 to about 15.0L/min/psid/ft²). With respect to plasma, a preferred permeability is inthe range of from about 0.00078 L/min/Pa/m² to about 0.0078 L/min/Pa/m²(about 0.5 to about 5.0 L/min/psid/ft²), more preferably in the range ofabout 0.0011 L/min/Pa/m² to about 0.0047 L/min/Pa/m² (about 0.7 to about3.0 L/min/psid/ft²).

The permeability and size of a typical separation device as provided bythe present invention is preferably sufficient to produce about 160 ccto about 240 cc of plasma at reasonable pressures (e.g., less than about6.9×10⁵ Pa (100 psi), more preferably, less than about 1.38×10⁵ (20 psi)in a reasonable amount of time (e.g., less than about one hour).

As provided by the present invention, all of these typical parametersmay be varied to achieve a desired result, e.g., varied preferably tominimize platelet loss, to maximize plasma-rich fluid production, and/orto establish a certain flow rate.

The separation device may be positioned in the system of the instantinvention in a variety of locations. For example, as illustrated in FIG.2, it may be located downstream of first container 19 and upstream ofsecond container 18 and third container 17 respectively. Alternatively,as illustrated in FIG. 1, it may be interposed between first container19 and second container 18.

A system as provided by the present invention may be used in conjunctionwith other functional biomedical devices, including filtration and/orseparation devices, e.g., a device for removing leukocytes from aplatelet-containing fluid or platelet concentrate. Exemplary functionalbiomedical devices are disclosed in U.S. Pat. Nos. 4,880,548, 4,925,572,and 5,100,564; and International Publication Nos. WO 92/07656 and WO91/17809. A functional biomedical device, as used herein, refers to anyof a number of devices, assemblies, or systems used in the collectionand/or processing of biological fluids, such as whole blood or a bloodcomponent. Exemplary functional biomedical devices include biologicalfluid containers, such as collection, transfer, and storage bags;conduits and connectors interposed between the containers; clamps,closures, and the like; air or gas inlet or outlet devices; a debubbler;a pump; and a red cell barrier device or assembly. The functionalbiomedical device may also include a device for destroying biologicalcontaminants, such as a high intensity light wave chamber, or a devicefor sampling a biological fluid.

The present inventive device may similarly be part of an apheresissystem. The biological fluid to be processed, the platelet-richsolution, and/or the platelet-poor solution may be handled in either abatch or continuous manner. The sizes, nature, and configuration of thepresent inventive device can be adjusted to vary the capacity of thedevice to suit its intended environment.

The processing of biological fluid in the context of the presentinvention may take place at any suitable time, which may be soon afterdonation. For example, when the biological fluid is donated whole blood,it is typically processed as soon as practicable in order to maximizethe number of components derived and to maximize blood componentviability and physiological activity. Early processing may moreeffectively reduce or eliminate contaminating factors, including, butnot limited to, leukocytes and microaggregates.

A method as provided by the invention may be described in more detailwith reference to FIGS. 1 and 2. Typically, a unit of a biologicalfluid, (e.g., donor's whole blood, or PRP) may be received into a firstcontainer 19 such as a collection bag or syringe for processing.

Movement of the biological fluid through the device and/or through thesystem may be effected by maintaining a pressure differential between acontainer such as a collection bag or a syringe containing thebiological fluid, and the destination of the biological fluid (e.g., acontainer such as a satellite bag), to cause the fluid to flow in adesired direction. Exemplary means for creating this pressuredifferential may be by gravity head, applying pressure to the container(e.g., by hand or with a pressure cuff), by placing the satellite bag ina chamber which establishes a pressure differential between thesatellite bag and the collection bag, e.g., a vacuum chamber or by apump. It is intended that the present invention is not to be limited bythe means of creating the pressure differential.

With reference to FIGS. 1 and 2, the biological fluid is processed bydirecting it from the container 19 to separation medium 16 so that thebiological fluid flows tangentially to the surface of the separationmedium. Directing the biological fluid to the separation medium mayinclude channelling the biological fluid tangentially to the surface ofthe separation medium such that a plasma-rich fluid passes tangentiallyacross the separation medium and a plasma-rich fluid passes through theseparation medium.

As noted above, establishing a tangential flow of the biological fluidbeing processed parallel with or tangential to the face of theseparation medium minimizes platelet collection within or passagethrough the separation medium. As provided by the invention, thetangential flow can be induced by any mechanical configuration of theflow path which induces a high local fluid velocity at the immediatemembrane surface. The tangential flow of the biological fluid may bedirected tangential or parallel to the face of the separation medium inany suitable manner, preferably utilizing a substantial portion of theseparation medium surface while maintaining a sufficient flow to ensurethat the platelets do not clog or block the pores of the separationmedium.

The flow of the biological fluid is preferably directed tangentially orparallel to the face of the separation medium through the use of atleast one fluid flow channel which is designed to maximize utilizationof the separation medium, ensure a sufficiently total area contactbetween the biological fluid and the separation medium, and maintain asufficient flow of biological fluid to minimize or prevent plateletadhesion to the separation medium. Most preferably, several (e.g., threeor more) uniform, serpentine, fluid flow channels are utilized so as toinduce a high local fluid velocity along the entire immediate membranesurface and to fix the separation medium in place and to prevent saggingof the membrane due to the applied pressure. In the embodimentsillustrated in FIGS. 3 and 6, three fluid flow channels are utilized inthe first flow path, and five fluid flow channels are utilized in thesecond fluid flow path.

The fluid flow channels may be of any suitable design and constructionand preferably are variable with respect to depth to maintain Optimalpressure and fluid flow across the face of the separation medium. Byproviding fluid flow channels, e.g., serpentine fluid flow channels, thebiological fluid flows through each channel at a velocity high enough tosweep clean the surface of the separation medium and prevent platelets,red cells, or other material from fouling the medium. By providingseveral channels, the velocity of the fluid is uniformly high across theentire surface of the separation medium. Consequently, no eddys orstagnant areas of the biological fluid develop where platelets, redcells, or other material may settle upon, stick to, and foul theseparation medium. Fluid flow channels may also be utilized on the sideof the separation medium opposite the biological fluid tangential flowto control the flow rate and pressure drop of a platelet-poor fluid,such as plasma.

In an exemplary method, the biological fluid enters inlet 11 of housing10 as shown in FIG. 2. From the inlet 11, the fluid enters the channels20-22 of the first chamber and passes tangentially or parallel to thefirst surface 16a of the separation medium 16 on the way to the firstoutlet 12 via the first fluid flow path 14. Plasma-rich fluid passesthrough the separation medium 16, and enters the channels 31-35 of thesecond chamber, and is directed toward the second outlet 13 via thesecond fluid flow path 15.

As the biological fluid continues along the first flow path 14tangentially or parallel to the first surface 16a of the separationmedium 16, more and more plasma-rich fluid crosses the separation medium16. A plasma-depleted fluid, e.g., a platelet-containing fluid, thenexits the housing 10 at the first outlet 12 while plasma-rich fluidexits the housing 10 at the second outlet 13. Typically, the plasma-richfluid may be stored in a region separated from the separation medium inorder to avoid possible reverse flow of the plasma-rich fluid backacross the separation medium to the plasma-depleted fluid.

The plasma-rich fluid exiting at the second outlet 13, and/or theplasma-depleted fluid exiting at the first outlet 12, may be furtherprocessed. For example, as shown in FIG. 2, additional processing mayinclude collecting the fluids in separate containers, such as firstsatellite bag 18 and second satellite bag 17. As shown in FIG. 1,additional processing may include re-directing the plasma-depleted fluidto the separation medium to deplete additional amounts of plasma. Theplasma-depleted fluid may be repeatedly recirculated through theseparation device, e.g., until the plasma-depleted fluid contains apre-determined amount or concentration of platelets.

The biological fluid may be supplied in any suitable quantity consistentwith the capacity of the overall device and by any suitable means, e.g.,in a batch operation by, for example, a blood bag connected to anexpressor or a syringe, or in a continuous operation as part of, forexample, an apheresis system.

In order that the invention herein described may be more fullyunderstood, the following examples are set out regarding use of thepresent invention. These examples are for illustrative purposes only andare not to be construed as limiting the present invention in any manner.

EXAMPLE 1

Whole blood was collected into an Adsol™ donor set and was processedunder standard conditions to yield a unit of PRP. The PRP was thenfiltered to remove leukocytes using a filter device described in U.S.Pat. No. 4,880,548. The removal efficiency was <99.9%.

The filtered PRP unit was then placed in a pressure cuff to which apressure of 300 mm Hg was applied. The tubing exiting the bag (clampedclosed at this point) was connected to the inlet port of a separationdevice as shown in FIGS. 3-6. A microporous polyamide membrane having apore rating of 0.65 microns was used as the separation medium in thedevice. The area of the membrane was about 17.4 square centimeters. Thedepth of the first fluid flow path channels decreased from about 0.03 cmnear the inlet to about 0.01 cm near the outlet. The depth of the secondfluid flow path channels was about 0.025 cm. The width of the channelswas 0.084 cm. The outlet ports of the device were connected to tubingwhich allowed the volume of fluid exiting the device to be measured andsaved for analysis.

The test of the present invention was started by opening the clamp andallowing PRP to enter the device. Clear fluid (plasma) was observed toexit one port, and turbid fluid (platelet concentrate) exited the otherport. The duration of the test was 42 minutes, during which 154 ml ofplasma and 32 ml of platelet concentrate was collected. Theconcentration of platelets in the plasma was found to be 1.2×10⁴ /μl,while the concentration of platelets in the platelet concentration wasfound to be 1.4333 10⁶ /μl.

The above results indicate that PRP can be concentrated to a usefullevel, and platelet-poor plasma recovered, in a reasonable time by adevice provided by the invention.

EXAMPLE 2

A sample of 450 ml of whole blood was collected under standardconditions from a human donor and placed in a typical flexible plasticblood bag. An analysis of the whole blood sample indicates that itcontained about 203 ml plasma. A 2 cc whole blood sample was withdrawnfrom the bag in a 5 cc syringe and attached to the inlet port of adevice as provided by the invention as shown in FIG. 2.

The present inventive device included a serpentine fluid flow path witha channel length of 32.5 cm, a constant width of 0.081 cm, and aconstant depth of 0.013 cm. The fluid flow path was of a "C"cross-section and, on its open side, contacted a microporouspolycarbonate membrane having a pore rating of 0.4 microns which servedas the separation medium. About 26.4 cm² of the microporous membranewere thereby part of the fluid flow path and were capable of beingcontacted by the whole blood sample or processed fluid as it passedthrough the device in the fluid flow path. Fluid flowed through theseparation medium at a rate of 0.2 ml/min. The entire whole blood samplewas processed in about 2 minutes. Air in the syringe was used to driveany hold-up through the device.

At the conclusion of the processing, a total of about 1.6 cc of turbidfluid (red cell containing fraction) and 0.4 cc of clear fluid wascollected from the processing of the whole blood sample. An analysis ofthe clear fluid indicated that it was plasma.

The above results indicate that plasma can be removed from whole bloodin a reasonable time through the use of the present invention.

EXAMPLE 3

A sample of 450 ml of whole blood is collected under standard conditionsfrom a human donor and placed in a typical flexible plastic blood bag.An analysis of the whole blood sample indicates that the hematocrit is37%, indicating that the sample includes about 283.5 cc plasma and 166.5cc red cells. A 2 cc whole blood sample is withdrawn from the bag in a 5cc syringe and attached to the inlet port of a device comprising aserpentine fluid flow path similar to that described in Example 2.

At the conclusion of the processing a total of about 0.75 cc of turbidfluid (red cell containing fraction) and 1.25 cc of clear fluid iscollected from the processing of the whole blood sample. An analysis ofthe clear fluid indicates that it is plasma.

The above results indicate that plasma can be efficiently removed fromwhole blood in a reasonable time through the use of the presentinvention.

EXAMPLE 4

A source bag and a satellite bag were connected to a separation deviceand a peristaltic pump in a configuration similar to that of FIG. 1. Thesource bag contained a unit of leukocyte depleted PRP (approximately 200ml). Tubing connected the source bag to the inlet port of the separationdevice, and, to provide for recirculation, tubing connected the firstoutlet port of the separation device to the source bag. Additionally,tubing connected the second outlet port of the separation device to thesatellite bag.

A peristaltic pump was associated with the tubing between the source bagand the inlet port of the separation device to provide for fluid flow.

The satellite bag was placed on a scale so that the amount of plasmaentering the bag could be monitored. The peristaltic pump was activatedat a flow rate of 25 cc/min and PRP was drawn from the source bag intothe device. Clear fluid (plasma) exited the second port and entered thesatellite bag. Turbid fluid (containing platelets) exited the first portand was recirculated into the source bag. The source bag wasperiodically squeezed to increase the mixing of the platelets in thefluid.

After approximately 35 minutes, about 150 ml of plasma was collected inthe satellite bag and about 50 ml of platelet concentrate was collectedin the source bag.

The above results indicate that platelet concentrate and platelet-poorplasma can be recovered in a reasonable time, using recirculation offluid by a device provided by the invention.

While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth. It should beunderstood that these specific embodiments are not intended to limit theinvention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

We claim:
 1. A method for processing a biological fluidcomprising:directing a biological fluid tangentially to the surface of aseparation medium in at least one serpentine fluid flow channel wherebyplasma-rich fluid passes through the separation medium andplasma-depleted fluid passes tangentially across the separation medium.2. The method of claim 1 further comprising recirculatingplasma-depleted fluid to the separation medium.
 3. The method of claim 1further comprising passing the plasma-rich fluid through at least oneflow channel.
 4. The method of claim 1 comprising passing the biologicalfluid through two or more serpentine fluid flow channels.
 5. A devicefor removing plasma from a biological fluid comprising:a housing havingan inlet and first and second outlets and defining a first fluid flowpath comprising at least one serpentine fluid flow channel between theinlet and the first outlet and a second fluid flow path between theinlet and the second outlet; and a separation medium positioned insidethe housing tangentially to the first fluid flow path and across thesecond fluid flow path, the separation medium being suitable for passingplasma therethrough.
 6. The device of claim 5 wherein said first fluidflow path comprises two or more serpentine fluid flow channels.
 7. Thedevice of claim 5 wherein said second fluid flow path comprises at leastone fluid flow channel.
 8. The device of claim 5 wherein said at leastone serpentine fluid flow channel is deeper near the inlet than near thefirst outlet.
 9. The device of claim 5 wherein said at least oneserpentine fluid flow channel decreases in depth over the length of thechannel in the fluid flow direction.
 10. The device of claim 7 whereinsaid fluid flow channel is a serpentine fluid flow channel.
 11. Thedevice of claim 10 wherein said second fluid flow path comprises two ormore serpentine fluid flow channels.
 12. A method for decreasing theplasma content of a biological fluid comprising:directing a biologicalfluid through the device of claim 8 tangentially to the surface of theseparation medium whereby a platelet-poor fluid passes through theseparation medium and a platelet-rich fluid is recovered.
 13. The methodof claim 12 further comprising directing the biological fluidtangentially to the surface of the separation medium at substantiallyconstant velocity.
 14. The method of claim 12 further comprisingrecirculating platelet-rich fluid through the device.